Mixotrophic, phototrophic, and heterotrophic combination methods and systems

ABSTRACT

Methods of culturing microorganisms in combinations of phototrophic, mixotrophic, and heterotrophic culture conditions are disclosed. A culture of microorganisms may be transitioned between culture conditions over the life of a culture in various combinations, utilizing various conditions in a sequential manner to optimize the culture for growth or product accumulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation International Application No.PCT/US2013/06049, filed Nov. 8, 2013 entitled Mixotrophic, Phototrophic,and Heterotrophic Combination Methods and Systems which claims thebenefit of U.S. Provisional Application No. 61/724,710, filed Nov. 9,2012, entitled Methods of Culturing Microorganisms in MixotrophicConditions; U.S. Provisional Application No. 61/798,969, filed Mar. 15,2013, entitled Mixotrophy Systems and Methods; and U.S. ProvisionalApplication No. 61/883,805, filed Sep. 27, 2013, entitled Mixotrophic,Phototrophic, and Heterotrophic Combination Methods and Systems, theentire contents of which are hereby incorporated by reference herein.

BACKGROUND

Microorganisms, such as microalgae and cyanobacteria, may be cultured ina variety of conditions that affect the microorganisms in multiple ways.With each set of culture conditions having unique advantages anddisadvantages, a culturing method using a single set of cultureconditions may not provide advantages in all the circumstances a cultureof microorganisms may experience over the life of the culture. In theprior art, methods of sequentially culturing microalgae in a firstphototrophic (or mixotrophic) stage and then a second heterotrophicstage are used to increase accumulation of medium length carbon chainlipids (e.g., C16:0) that are used for biofuel production. Thesequential stages of culture conditions attempt to leverage the low costof phototrophic methods with the high productivity of heterotrophicmethods to increase the efficiency of the biofuel production process.While this sequence may be beneficial for the production of lipids usedin biofuels, the method is not optimized for the variety of diverseproduct markets that may be served by microalgae and cyanobacteriathrough whole biomass, proteins, pigments, polysaccharides, nutritionalfatty acids, and others microorganism products.

Beyond lipid production for biofuels, the culture conditions may be morebroadly applicable to enhancing the rate of growth, controlling thelevel of contaminating organisms (e.g., contaminating bacteria,competing organisms, fungi), product formation and accumulation (e.g.,proteins, lipids, pigments), and other aspects relevant to thecommercial production of microorganisms. Mixotrophic microorganismsprovide the capability to culture microorganisms in a more flexiblesystem utilizing a variety of culture conditions for different purposesor for different functions of the microorganisms. Therefore, there is aneed in the art for flexible methods of culturing mixotrophicmicroorganisms in a plurality of culture conditions which optimize theculture for growth and a diverse product profile.

SUMMARY

Embodiments described herein relate generally to methods for culturingmicroorganisms in combinations of phototrophic, mixotrophic, andheterotrophic culture conditions. A culture of microorganisms may betransitioned between culture conditions over the life of a culture,utilizing various conditions in a sequential manner to optimize theculture for growth or product accumulation.

In some embodiments of the invention, a method of culturingmicroorganisms comprises: providing a culture of microorganisms capableof growth in mixotrophic and phototrophic culture conditions; growingthe culture of microorganisms in mixotrophic culture conditions; growingthe culture of microorganisms in phototrophic culture conditions absenta supply of the organic carbon source; and transitioning the culture ofmicroorganisms between mixotrophic and phototrophic conditions at leastonce in the life of the culture of microorganisms. The mixotrophicculture conditions comprise: supplying the culture of microorganismswith at least some light; supplying the culture of microorganisms withat least one organic carbon source; and supplying the culture ofmicroorganisms with carbon dioxide. The phototrophic culture conditionscomprise: supplying the culture of microorganisms with light and carbondioxide.

In some embodiments, the microorganisms are selected from the groupconsisting of microalgae and cyanobacteria. In some embodiments, the atleast one organic carbon source is selected from the group consisting ofacetate, acetic acid, ammonium linoleate, arabinose, arginine, asparticacid, butyric acid, cellulose, citric acid, ethanol, fructose, fattyacids, galactose, glucose, glycerol, glycine, lactic acid, lactose,maleic acid, maltose, mannose, methanol, molasses, peptone, plant basedhydrolyzate, proline, propionic acid, ribose, sacchrose, partial orcomplete hydrolysates of starch, sucrose, tartaric, TCA-cycle organicacids, thin stillage, urea, industrial waste solutions, and yeastextracts.

In some embodiments, transitioning the culture of microorganisms betweenmixotrophic and phototrophic conditions at least once in the life of theculture of microorganisms comprises harvesting a portion of the cultureof microorganisms in mixotrophic conditions to inoculate the harvestedculture of microorganisms in phototrophic conditions. In someembodiments, transitioning the culture of microorganisms betweenmixotrophic and phototrophic conditions at least once in the life of theculture of microorganisms comprises harvesting a portion of the cultureof microorganisms in phototrophic conditions to inoculate the harvestedculture of microorganisms in mixotrophic conditions.

In some embodiments, transitioning the culture of microorganisms betweenmixotrophic and phototrophic conditions at least once in the life of theculture of microorganisms comprises at least one of decreasing thechlorophyll content, decreasing the sterols content, decreasing thetotal amino acid content, increasing the carotene content, andincreasing the carbohydrate content in the culture of microorganisms bytransitioning the culture of microorganisms from phototrophic tomixotrophic conditions before harvest. In some embodiments,transitioning the culture of microorganisms between mixotrophic andphototrophic conditions at least once in the life of the culture ofmicroorganisms comprises at least one of increasing the chlorophyllcontent, increasing the sterols content, increasing the total amino acidcontent, decreasing the carotene content, and decreasing thecarbohydrate content in the culture of microorganisms by transitioningthe culture of microorganism from mixotrophic to phototrophic conditionsbefore harvest.

In some embodiments of the invention, a method of culturingmicroorganisms comprises: providing a culture of microorganisms in anaqueous medium capable of growth on light as an energy source, carbondioxide as a carbon source, and at least one organic carbon source as anenergy and carbon source; growing the culture of microorganisms inphototrophic conditions, wherein the phototrophic conditions comprise asupply of light as an energy source and carbon dioxide as a carbonsource in a phototrophic culturing vessel comprising a volume less than10 L and a lack of a supply of at least one organic carbon source;harvesting at least a portion of the culture of microorganisms from thephototrophic culturing vessel; inoculating the culture of microorganismsharvested from the phototrophic culturing vessel into a small volumemixotrophic culturing vessel, wherein the small volume mixotrophicculturing vessel comprises a volume of 10 to 100 L and operates in amixotrophic conditions for growing the culture of microorganisms,wherein the mixotrophic conditions comprise a supply of light as anenergy source, carbon dioxide as an inorganic carbon source, and atleast one organic carbon source as an energy and carbon source;harvesting at least a portion of the culture of microorganisms form thesmall volume mixotrophic culturing vessel; inoculating the culture ofmicroorganisms harvested from the small volume mixotrophic culturingvessel into a large volume mixotrophic culturing vessel, wherein thelarge volume mixotrophic culturing vessel comprises a volume of at least100 L and operates in mixotrophic conditions for growing the culture ofmicroorganisms; and harvesting the culture of microorganisms from thelarge volume mixotrophic culturing vessel.

In some embodiments, the phototrophic culturing vessel is closed oropen. In some embodiments, the small volume mixotrophic culturing vesselis closed or open. In some embodiments, the large volume mixotrophicculturing vessel is closed or open. In some embodiments, the largevolume mixotrophic culturing vessel comprises a volume of 100 to 750,000L.

In some embodiments of the invention, a method of culturingmicroorganisms comprises: providing a culture of microorganisms in anaqueous culture medium capable of growth heterotrophic, mixotrophic, andphototrophic culture conditions; disposing the culture of microorganismsin non-axenic conditions in a culturing vessel wherein the culture ofmicroorganisms is exposed to at least some sunlight during daylighthours and no sunlight during night hours, the daylight hours comprisingpeak hours of sunlight and non-peak hours of sunlight; supplying atleast one organic carbon source to the culture of microorganisms duringa time period comprising the non-peak hours of sunlight and night hours,the peak hours of sunlight comprising an equal amount of time on eitherside of a time when the sun is at its zenith and non-peak hours ofsunlight comprising all other hours of sunlight during daylight hours;eliminating the supply of at least one organic carbon source during thepeak hours of sunlight; and growing the culture of microorganisms for atleast two consecutive days.

In some embodiments, the peak hours of sunlight are 6-10 hours. In someembodiments, growing the culture of microorganisms is continued for 2 to90 consecutive days. In some embodiments, the method further comprisessupplying carbon dioxide to the culture of microorganisms during peakhours of sunlight and non-peak hours of sunlight. In some embodiments,the method further comprises detecting the level of sunlight with aphotodetector and controlling the supply of organic carbon with aprogrammable logic controller which receives data from thephotodetector.

In some embodiments, a method of culturing microorganisms comprises:providing a culture of microorganisms in an aqueous medium capable ofgrowth on light as an energy source, carbon dioxide as a carbon source,and at least one organic carbon source as an energy and carbon source;transitioning the culture of microorganisms between at least twoselected from the group consisting of phototrophic, mixotrophic andheterotrophic culture conditions, at least once in the life of theculture of microalgae; and wherein the transition between at least twoselected from the group consisting of phototrophic, mixotrophic, andheterotrophic culture conditions occurs when at least one thresholdcondition of the culture it met.

The phototrophic culture conditions comprise supplying the culture ofmicroorganisms with light and carbon dioxide in the absence of a supplyof an organic carbon source. The mixotrophic culture conditions comprisesupplying the culture of microorganisms with light, carbon dioxide, andthe at least one organic carbon source. The heterotrophic cultureconditions comprise supplying the culture of microorganisms with the atleast one organic carbon source in the absence of a supply of light andcarbon dioxide. The threshold conditions of the culture comprise: adetected level of dissolved oxygen, a detected level of dissolved carbondioxide, a detected level of contaminating organisms, a detected levelof chlorophyll, a detected temperature level, a detected residualorganic carbon level, and complete consumption of a batch of organiccarbon.

In some embodiments, the contaminating organisms comprise bacteria orfungi. In some embodiments, the culture of microorganisms transitionsfrom phototrophic to mixotrophic culture conditions when the detecteddissolved oxygen level is above a threshold level. In some embodiments,the culture of microorganisms transitions from phototrophic toheterotrophic culture conditions when the detected dissolved oxygenlevel is above a threshold level. In some embodiments, the culture ofmicroorganisms transitions from mixotrophic to phototrophic cultureconditions when the detected dissolved oxygen level is below a thresholdlevel. In some embodiments, the culture of microorganisms transitionsfrom heterotrophic to phototrophic culture conditions when the detecteddissolved oxygen level is below a threshold level.

In some embodiments, the culture of microorganisms transitions frommixotrophic to phototrophic culture conditions when the detecteddissolved carbon dioxide level is above a threshold level. In someembodiments, the culture of microorganisms transitions fromheterotrophic to phototrophic culture conditions when the detecteddissolved carbon dioxide level is above a threshold level. In someembodiments, the culture of microorganisms transitions from phototrophicto mixotrophic culture conditions when the detected dissolved carbondioxide level is below a threshold level. In some embodiments, theculture of microorganisms transitions from phototrophic to heterotrophicculture conditions when the detected dissolved carbon dioxide level isbelow a threshold level.

In some embodiments, the culture of microorganisms transitions fromheterotrophic to mixotrophic culture conditions when the detectedchlorophyll level is below a threshold level. In some embodiments, theculture of microorganisms transitions from heterotrophic to phototrophicculture conditions when the detected chlorophyll level is below athreshold level. In some embodiments, the culture of microorganismstransitions from mixotrophic to phototrophic culture conditions when thedetected chlorophyll level is below a threshold level. In someembodiments, the culture of microorganisms transitions from phototrophicto mixotrophic culture conditions when the detected chlorophyll level isabove a threshold level. In some embodiments, the culture ofmicroorganisms transitions from phototrophic to heterotrophic cultureconditions when the detected chlorophyll level is above a thresholdlevel.

In some embodiments, the method further comprises measuring density ofthe culture and the culture of microorganisms transitions fromphototrophic to mixotrophic culture conditions when the detected cellculture density level is above a first threshold level. In someembodiments, the culture of microorganisms transitions from mixotrophicto heterotrophic culture conditions when the detected cell culturedensity level is above a second threshold level.

In some embodiments, the culture of microorganisms transitions fromphototrophic to mixotrophic culture conditions when the detected levelof contaminating organisms is below a threshold level. In someembodiments, the culture of microorganisms transitions from phototrophicto heterotrophic culture conditions when the detected level ofcontaminating organisms is below a threshold level. In some embodiments,the culture of microorganisms transitions from mixotrophic tophototrophic culture conditions when the detected level of contaminatingorganisms is above a threshold level. In some embodiments, the cultureof microorganisms transitions from heterotrophic to phototrophic cultureconditions when the detected level of contaminating organisms is above athreshold level.

In some embodiments, the culture of microorganisms transitions fromphototrophic to mixotrophic culture conditions when the detected levelof residual organic carbon is below a threshold level. In someembodiments, the culture of microorganisms transitions from phototrophicto heterotrophic culture conditions when the detected level of residualorganic carbon is below a threshold level.

In some embodiments, the culture of microorganisms transitions fromheterotrophic to phototrophic culture conditions when the detectedculture temperature is below a threshold level. In some embodiments, theculture of microorganisms transitions from mixotrophic to phototrophicculture conditions when the detected culture temperature is below athreshold level. In some embodiments, the culture of microorganismstransitions from phototrophic to mixotrophic culture conditions when thedetected culture temperature is above a threshold level. In someembodiments, the culture of microorganisms transitions from phototrophicto heterotrophic culture conditions when the detected culturetemperature is above a threshold level.

In some embodiments, the culture of microorganisms transitions fromheterotrophic to phototrophic culture conditions upon the completeconsumption of a batch of organic carbon. In some embodiments, theculture of microorganisms transitions from mixotrophic to phototrophicculture conditions upon the complete consumption of a batch of organiccarbon.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing the product profile of microalgae grown inphototrophic and mixotrophic conditions.

FIG. 2 is a graph comparing the product profile of microalgae grown inphototrophic and mixotrophic conditions.

FIG. 3 is a graph comparing the fatty acid profile of microalgae grownin phototrophic and mixotrophic conditions.

FIG. 4 shows an embodiment of a fermentation vessel.

FIG. 5 is a graph showing the growth rate of Haematococcus inheterotrophic growth conditions.

FIG. 6 is a graph showing the ratio of red and green cysts in a cultureof Haematococcus.

DETAILED DESCRIPTION Definitions

The term “microorganism” refers to microscopic organisms such asmicroalgae and cyanobacteria. Microalgae include microscopicmulti-cellular plants (e.g. duckweed), photosynthetic microorganisms,heterotrophic microorganisms, diatoms, dinoflagelattes, and unicellularalgae.

The terms “microbiological culture”, “microbial culture”, or“microorganism culture” refer to a method or system for multiplyingmicroorganisms through reproduction in a predetermined culture medium,including under controlled laboratory conditions. Microbiologicalcultures, microbial cultures, and microorganism cultures are used tomultiply the organism, to determine the type of organism, or theabundance of the organism in the sample being tested. In liquid culturemedium, the term microbiological, microbial, or microorganism culturegenerally refers to the entire liquid medium and the microorganisms inthe liquid medium regardless of the vessel in which the culture resides.A liquid medium is often referred to as “media”, “culture medium”, or“culture media”. The act of culturing is generally referred to as“culturing microorganisms” when emphasis is on plural microorganisms.The act of culturing is generally referred to as “culturing amicroorganism” when importance is placed on a species or genus ofmicroorganism. Microorganism culture is used synonymously with cultureof microorganisms.

Microorganisms that may grow in mixotrophic culture conditions includemicroalgae, diatoms, and cyanobacteria. Non-limiting examples ofmixotrophic microorganisms may comprise organisms of the genera:Agmenellum, Amphora, Anabaena, Anacystis, Apistonema, Pleurochyrsis,Arthrospira (Spirulina), Botryococcus, Brachiomonas, Chlamydomonas,Chlorella, Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris,Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena, Extubocellulus,Fragilaria, Galdieria, Goniotrichium, Haematococcus, Halochlorella,Isochyrsis, Leptocylindrus, Micractinium, Melosira, Monodus, Nostoc,Nannochloris, Nannochloropsis, Navicula, Neospongiococcum, Nitzschia.,Odontella, Ochromonas, Ochrosphaera, Pavlova, Picochlorum,Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas,Prymnesium, Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis,Stichococcus, Auxenochlorella, Cheatoceros, Neochloris, Ocromonas,Porphiridium, Synechococcus, Synechocystis, Tetraselmis,Thraustochytrids, Thalassiosira, and species thereof.

The organic carbon sources suitable for growing a microorganismmixotrophically or heterotrophically may comprise: acetate, acetic acid,ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid,cellulose, citric acid, ethanol, fructose, fatty acids, galactose,glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose,mannose, methanol, molasses, peptone, plant based hydrolyzate, proline,propionic acid, ribose, sacchrose, partial or complete hydrolysates ofstarch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea,industrial waste solutions, yeast extract, and combinations thereof. Theorganic carbon source may comprise any single source, combination ofsources, and dilutions of single sources or combinations of sources.

The terms “mixotrophic” and “mixotrophy” refer to culture conditions inwhich light, organic carbon, and inorganic carbon (e.g., carbon dioxide,carbonate, bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in mixotrophic conditions have themetabolic profile of both phototrophic and heterotrophic microorganisms,and may use both light and organic carbon as energy sources, as well asboth inorganic carbon and organic carbon as carbon sources. Amixotrophic microorganism may be using light, inorganic carbon, andorganic carbon through the phototrophic and heterotrophic metabolismssimultaneously or may switch between the utilization of each metabolism.A microorganism in mixotrophic culture conditions may be a net oxygen orcarbon dioxide producer depending on the energy source and carbon sourceutilized by the microorganism. Microorganisms capable of mixotrophicgrowth comprise microorganisms with the natural metabolism and abilityto grow in mixotrophic conditions, as well as microorganisms whichobtain the metabolism and ability through modification of cells by wayof methods such as mutagenesis or genetic engineering.

The terms “phototrophic”, “phototrophy”, “photoautotrophy”,“photoautotrophic”, and “autotroph” refer to culture conditions in whichlight and inorganic carbon (e.g., carbon dioxide, carbonate,bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in phototrophic conditions may uselight as an energy source and inorganic carbon (e.g., carbon dioxide) asa carbon source. A microorganism in phototrophic conditions may produceoxygen.

The terms “heterotrophic” and “heterotrophy” refer to culture conditionsin which organic carbon may be applied to a culture of microorganisms inthe absence of light. Microorganisms capable of growing in heterotrophicconditions may use organic carbon as both an energy source and as acarbon source. A microorganism in heterotrophic conditions may producecarbon dioxide.

The term “axenic” describes a culture of an organism that is entirelyfree of all other “contaminating” organisms (i.e., organisms that aredetrimental to the health of the microalgae or cyanobacteria culture).Throughout the specification, axenic refers to a culture that wheninoculated in an agar plate with bacterial basal medium, does not formany colonies other than the microorganism of interest. Axenic describescultures not contaminated by or associated with any other livingorganisms such as but not limited to bacteria, cyanobacteria, microalgaeand/or fungi. Axenic is usually used in reference to pure cultures ofmicroorganisms that are completely free of the presence of otherdifferent organisms. An axenic culture of microalgae or cyanobacteria iscompletely free from other different organisms.

Bacteria that may be present in cultures of microalgae and cyanobacteriacomprise, but are not limited to: Achromobacter sp., Acidovorax sp.,Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp.,Ancylobacter sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp.,Bacillus sp., Bergeyella sp., Brevundimonas sp., Brochothrix sp.,Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp.,Chryseobacterium sp., Curtobacterium sp., Delftia sp., Empedobacter sp.,Enterobacter sp., Escherichia sp., Flavobacterium sp., Gemmatimonas sp.,Halomonas sp., Hydrogenophaga sp., Janthinobacterium sp., Lactobacillussp., Marinobacter sp., Massilia sp., Microbacterium sp., Myroides sp.,Pantoea sp., Paracoccus sp., Pedobacter sp., Phaeobacter sp.,Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonas sp., Rahnellasp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp.,Sphingobacterium sp., Sphingomoas sp., Staphylococcus sp.,Stenotrophomonas sp., Vibrio sp., and Zobelliae sp.

Bacteria that have a negative or harmful effect on the microalgae andcyanobacteria may be designated as contaminating bacteria. The bacteriathat may have a negative or harmful effect on microalgae orcyanobacteria in a culture comprise, but are not limited to:Achromobacter sp., Acidovorax sp., Aeromonas sp., Agrobacterium sp.,Alteromonas sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp.,Bergeyella sp., Brochothrix sp., Brumimicrobium sp., Burkholderia sp.,Caulobacter sp., Cellulomonas sp., Chryseobacterium sp., Curtobacteriumsp., Delftia sp., Empedobacter sp., Enterobacter sp., Escherichia sp.,Flavobacterium sp., Marinobacter sp., Microbacterium sp., Myroides sp.,Paracoccus sp., Pedobacter sp., Phaeobacter sp., Pseudoalteromonas sp.,Pseudomonas sp., Rahnella sp., Ralstonia sp., Rhizobium sp., Rhodococcussp., Roseomonas sp., Staphylococcus sp., Stenotrophomonas sp., Vibriosp., Zobelliae sp. and other bacteria which share similarcharacteristics.

The bacteria that may have a neutral or beneficial effect on microalgaeor cyanobacteria in a culture comprise, but are not limited to:Acidovorax sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp.,Alteromonas sp., Ancylobacter sp., Azospirillum sp., Azotobacter sp.,Bacillus sp., Brevundimonas sp., Brumimicrobium sp., Burkholderia sp.,Caulobacter sp., Cellulomonas sp., Delftia sp., Empedobacter sp.,Gemmatimonas sp., Halomonas sp., Hydrogenophaga sp., Janthinobacteriumsp., Lactobacillus sp., Marinobacter sp., Pantoea sp., Paracoccus sp.,Phaeobacter sp., Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonassp., Rhizobium sp., Sphingomoas sp., Zobelliae sp. and other bacteriawhich share similar characteristics. While bacteria in a particulargenus generally have the same characteristics, it is recognized that agenus of bacteria with the majority of species generally identified asharmful to microalgae or cyanobacteria may also include a particularspecies within the genus which is neutral or beneficial to a specificculture of microalgae or cyanobacteria, and vice versa. For example,many species of Pseudomonas have been observed to be harmful tomicroalgae, however literature has described certain species ofPseudomonas with anti-fungal functionality which may be beneficial to aculture of microalgae or cyanobacteria.

The term “pH auxostat” refers to the microbial cultivation techniquethat couples the addition of fresh medium (e.g., medium containingorganic carbon or acetic acid) to pH control. As the pH drifts from agiven set point, fresh medium is added to bring the pH back to the setpoint. The rate of pH change is often an excellent indication of growthand meets the requirements as a growth-dependent parameter. The feedwill keep the residual nutrient concentration in balance with thebuffering capacity of the medium. The pH set point may be changeddepending on the microorganisms present in the culture at the time. Themicroorganisms present may be driven by the location and season wherethe bioreactor is operated and how close the cultures are positioned toother contamination sources (e.g., other farms, agriculture, ocean,lake, river, waste water). The rate of medium addition is determined bythe buffering capacity and the feed concentration of the limitingnutrient and not directly by the set point (pH) as in a traditionalauxostat. The pH auxostat is robust but controls nutrient concentrationindirectly. The pH level represents the summation of the production ofdifferent ionic species and ion release during carbon and nutrientuptake. Therefore the pH level can move either up or down as a functionof growth of the microorganisms. The most common situation is pHdepression caused by organic acid production and ammonium uptake.However, for microorganisms growing on protein or amino acid-rich media,the pH level will rise with growth because of the release of excessammonia.

The term “harvesting” refers to removing the culture of microorganismsfrom the culturing vessel and/or separating the microorganisms from theculture medium. Harvesting of microorganisms may be conducted by anymethod known in the art such as, but not limited to, skimming, draining,dissolved gas flotation, foam fractionation, centrifugation, filtration,sedimentation, chemical flocculation, and electro-dewatering.

The term “inoculate” refers to implanting or introducing microorganismsinto a culture medium. Inoculate or inoculating a culture ofmicroorganisms in the described culture conditions throughout thespecification refers to starting a culture of microorganisms in theculture conditions, as is commonly used in the art of microorganismculturing. The microorganisms that are introduced into a culture mediummay be referred to as seed or inoculum.

Overview

Cultures of mixotrophic microorganisms, such as microalgae andcyanobacteria, may be grown solely in mixotrophic conditions receivingan organic carbon supply, an optional inorganic carbon supply (e.g.,carbon dioxide, carbonate, bi-carbonate), and at least some light(either intermittent or continuous) from the beginning to end of thelife of the culture of the mixotrophic microorganisms. Whilemicroorganism cultures may experience growth benefits in a mixotrophiconly system, the selection of culture conditions may be reversible andflexible over the life of the culture allowing for the use of a varietyof different culture conditions. Such a transition between cultureconditions, such as phototrophic, mixotrophic, and heterotrophic, mayprovide benefits such as, but not limited to, an increase in overallmicroorganism growth, an increase in initial microorganism growth,manipulation of product formation in the microorganisms, an extension tothe life of the culture of microorganisms, and the suppression ofcontaminating organisms (e.g., bacteria, fungi, competing organisms,invading species of microalgae) in a culture. Phototrophic, mixotrophic,and heterotrophic culturing conditions may be conducted in any suitableculturing vessel or bioreactor such as, but not limited to, a pond, araceway pond, a trough, a tank, a tubular bioreactor, a flat panelbioreactor, a bag bioreactor, and a column bioreactor.

For example, methods for cultures of microorganisms with transitionsbetween trophic conditions may regulate the feed source forcontaminating bacteria (i.e., bacteria which have a negative or harmfuleffect on a culture of microalgae or cyanobacteria), while allowing thedesired microorganisms to continue to grow in the varied conditions.Microorganisms, such as microalgae and cyanobacteria, growth may slowdown in phototrophic conditions compared to mixotrophic or heterotrophicconditions, but the contaminating bacteria growth may slow down as welland possibly stop if the bacteria does not have a photosyntheticmetabolism. Additionally, microorganism culturing methods withtransitions between trophic conditions may regulate the dissolved oxygenlevel in the microorganism culture, which may negatively affectcontaminating bacteria while allowing the microorganisms to continue togrow in the varied conditions. When dissolved oxygen concentrationsreach the targeted concentrations and the contaminating bacteriapopulations have been reduced, the system may be transitioned fromphototrophic conditions back to mixotrophic conditions thus creating acyclic pattern that reduces contaminating bacteria and increasesmicroalgae or cyanobacteria culture longevity.

In product formation, microorganisms may provide a variety of proteins,polysaccharides, lipids, and pigments that are influenced by the cultureconditions. The formation of products may be influenced by the energyinputs, carbon inputs, temperature, pH, amount of growth, or stage ofgrowth provided by and dictated by different culture conditions.

Transitions Between Phototrophy and Mixotrophy

Cultures of microorganisms may be transitioned between mixotrophy andphototrophy by controlling the supply of the organic carbon, light, andinorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate). Amixotrophic culture of microorganisms, such as microalgae andcyanobacteria, may provide inoculum to a phototrophic culture bytransferring a portion of the microorganisms harvested from amixotrophic culture to a culture medium and system for phototrophicgrowth. Growing a microorganism mixotrophically first allows the cultureto take advantage of the boost in initial growth experienced inmixotrophic conditions to provide a sufficient volume of seed orinoculum for a plurality of phototrophic cultures. The transitionbetween culturing methods may also go from phototrophic to mixotrophicconditions, allowing lower performing cultures in phototrophicconditions to receive a boost in productivity through conversion tohigher performing mixotrophic conditions that provide both organiccarbon and light as energy sources.

Additionally, transitioning a culture between phototrophic andmixotrophic culturing conditions, and vice versa, may also affect theproduct formation in the microorganism. Pigments (e.g., chlorophyll,carotenoids, phycocyanin), polysaccharides, proteins, amino acids,hormones, lipids, fatty acids, carbohydrates and other products mayaccumulate differently in the microorganism depending on the cultureconditions. Also, a culture growing mixotrophically may be converted tophototrophic growth to allow for photopigment saturation in themicroorganisms, leading to increased product formation and/ormicroorganism health. Therefore control over the microorganism productsmay be exercised by the choice of culturing conditions and/or the timingof the transition between culturing conditions.

In another embodiment, the level of contaminating organisms (e.g.,bacteria, fungi, completing organisms, invading species of microalgae)in a culture of microorganisms may be managed by transitioning betweenmixotrophy and phototrophy. In some embodiments, the culture ofmicroorganisms may be transitioned from mixotrophy to phototrophy for aperiod of time (e.g., 24-48 hours), and a harvest may occur whencultures are transitioned to reduce the total organic carbon content inthe culture. By reducing the total organic carbon in the culture, theavailable feed sources for contaminating organisms may be reduced andaid in controlling contaminating populations. The transition frommixotrophy to phototrophy itself may also reduce the organic carbonsource in the culture medium without a harvest, and reduce the feedavailable to contaminating organisms.

Example 1

Samples of a Chlorella species were obtained from phototrophic andmixotrophic open pond cultures (pond volume of 570 L) and compared.These samples were harvested by centrifuging, dried using a freeze drierand analyzed for the composition of the algae at POS Bio-Sciences(Saskatoon, Canada). The phototrophically cultured Chlorella was grownunder non-axenic conditions using standard procedures known in the artcomprising an f/2 culture medium with trace metals, a culturetemperature of approximately 28° C., a raceway pond photobioreactor, asupply of sunlight, sparging of air within the culture medium, andsparging of carbon dioxide within the culture medium.

The mixotrophically cultured Chlorella was grown under non-axenicconditions using an acetic acid/pH auxostat feeding system to supplyorganic carbon in non-axenic conditions for 10 days. The trial wasperformed with two raceway pond photobioreactors made of PVC, with acultivable area of 5.6 m² and a 10 cm light path (i.e., distance oflight penetration). Both photobioreactors contained mixotrophic culturesaerated with two 50 cm porous diffusers at 10 liters per minute (LPM)and were located outdoors. The first photobioreactor (Reactor 1) wasmixed hydraulically (pump). The second photobioreactor (Reactor 2) wasmixed with a paddle wheel. The Chlorella was inoculated at a density of0.3 g/L in the Reactors 1 and 2. The Chlorella was adapted to theoutdoor conditions under CO₂/pH control until it attained a density of0.3-0.4 g/L for the experimental trials. The acetic acid supply occurredusing the pH auxotstat drip system which dosed acetic acid when thedetected pH level of the mixotrophic cultures was above the set point of7.5.

The mixotrophic cultures were harvested as needed when the culturedensity reached 1.5 g/L. The initial mixotrophic culture medium was aBG-11 medium with sodium acetate (1 g/L) supplemented in the initialmedium. Natural sunlight was supplied to the mixotrophic cultures, withthe average photoperiod in May 2012 for Gilbert, Ariz. being about 14.5h. The temperature was controlled by cooling coils at 28° C. The pHcontroller of the pH auxostat system, as previously described, was setat 7.5. Temperature, pH and dissolved oxygen were measured continuously.Acetic acid consumption was monitored by the acetic acid feed tank leveldaily. Dry weights were taken three times (n=3) daily and nitrate levelswere taken daily. Spin down to measure residual acetate (200 ml) andbiomass was performed every two days.

Contamination observation (400×, 1000× oil immersion-phase contrastmicrograph and cell cytometry with bacterial dying) was performed everytwo days, including a measurement of bacterial contamination by flowcytometry. For bacterial contamination measurement, to each 1 ml sample,1 μl BacLight™ Green bacterial stain (Invitrogen, Eugene, Oreg., USA)was added, and samples were incubated at room temperature in the darkfor 30 to 60 minutes. After incubation, samples were analyzed on a BDFACSAria™ (BD Biosciences, San Jose, Calif., USA) and populations ofbacteria and microalgae were gated based on BacLight™ fluorescence andchlorophyll autofluoresence.

FIGS. 1-3 compare the compositions of the Chlorella produced inmixotrophic and phototrophic cultures as determined by POS analyticalmethods, and illustrate the differences in product formation that resultfrom the two culturing methods. Referring to FIG. 1, the fat percent ofthe mixotrophically cultured microalgae was about 13% of dry weight,while the phototrophically cultured microalgae was approximately 2.6times lower at about 5%. The carbohydrates percent showed similarresults with the mixotrophic microalgae comprising about 48% and thephototrophic microalgae being about 1.4 times lower at about 35%. Theprotein accumulation represented by the total amino acids content in dryweight was lower in the mixotrophically cultured microalgae.

FIG. 2 compares additional high value components in the Chlorellasamples. FIG. 2 shows that the phototrophically cultured microalgae hadabout 12 times more chlorophyll content than the mixotrophicallycultured microalgae. The xanthophylls and tocopherols were slightlyhigher in the mixotrophic culture than the phototrophic culture.

As shown in the fatty acid composition comparison of FIG. 3,concentration of monounsaturated fatty acids almost tripled from about6% of total fatty acid in the phototrophic culture to about 16% in themixotrophic culture. Particularly, the oleic acid concentrationincreased significantly from about 2% for the phototrophic culture toabout 14% for the mixotrophic culture. However, the saturates alsodoubled in the phototrophic culture as compared to the mixotrophicculture, specifically palmitic acid increased from about 8% in thephototrophic culture to about 19% in the mixotrophic culture. Theconcentration of palmitoleic acid was slightly higher in thephototrophically cultured microalgae than in the mixotrophicallycultured microalgae. Also, the palmitic acid concentration was much morethan the palmitoleic acid concentration, whereas the stearic acid wasalmost negligible compared to the oleic acid and polyunsaturated formsin the mixotrophically cultured microalgae. Overall, the microalgaecultured in mixotrophic conditions may be more valuable as a food orfuel precursor due to higher concentration of unsaturated fatty acids(for food, nutraceutical applications) and a high concentration ofpalmitic acid (for fuel applications).

Several key deductions may be made from the compositional analysis: 1)as observed in the example, the mixotrophic conditions provide theadvantage of producing high levels of carotenes (similar to aphototrophic culture), and high growth rates (similar to a heterotrophicculture); and 2) the ability to transition from phototrophic tomixotrophic culturing conditions and vice versa, provides flexibility inthe product profile of the microalgae. For example, the chlorophyllcontent may be reduced significantly by transitioning from phototrophicto mixotrophic culture conditions just before harvest, which neutralizesthe product coloring. Such neutralization of product coloring may bedesirable in food and feed applications. Transitioning the culturebetween phototrophy and mixotrophy may similarly increase or decreasethe product content as demonstrated in FIGS. 1-3.

Multi-Stage Culturing with Phototrophy and Mixotrophy

In some embodiments, a multi-stage commercial method of culturingmicroorganisms, such as microalgae and cyanobacteria, may comprise atransition between culturing conditions in combination with multiplestages in a single culture condition. In some embodiments, a commercialmethod may have different culture volumes for different cultureconditions at different stages. In some embodiments, a commercial methodmay have different culture volumes for different stages of the sameculture conditions.

In some embodiments, a commercial method of culturing microorganisms maycomprise growing a culture of microorganisms in phototrophic conditionsin a first stage, and growing the culture of microorganisms inmixotrophic conditions in at least one subsequent stage. In a preferredembodiment, the subsequent stages in mixotrophic conditions may compriseat least two stages. In some embodiments, the phototrophic conditions ofthe first stage comprise a phototrophic culturing vessel with a volumeless than 10 L. In some embodiments, the culture of microorganisms maybe harvested from the phototrophic culturing vessel and used toinoculate a small volume mixotrophic culturing vessel comprising avolume of 10 to 100 L in a culturing stage using mixotrophic conditions.In some embodiments, the culture of microorganisms may be harvested fromthe small volume mixotrophic culturing vessel and used to inoculate alarge volume mixotrophic culturing vessel comprising a volume greaterthan 100 L in a culturing stage using mixotrophic conditions. In someembodiments, the large volume mixotrophic culturing vessel comprises avolume between 100 and 750,000 L. The culture of microorganisms may beharvested from the large volume mixotrophic culturing vessel fordownstream processing such as, but not limited to, dewatering,extraction, esterification, transesterification, hydrotreatment. In someembodiments, the culturing vessels may be open culturing vessels suchas, but not limited to, troughs, ponds and raceway ponds. In someembodiments, the culturing vessels may be closed culturing vessels suchas, but not limited to, tanks, column bioreactors, bag bioreactors,column bioreactors, and tubular bioreactors.

In some embodiments, a culture of microorganisms may be culturedmixotrophically for 3-14 days before harvesting the entire volume, andthe harvested volume may be split among up to 10 bioreactors of equalvolume (i.e., essentially diluting each culture by up to 90%). Theplurality of bioreactors may culture the microorganisms in phototrophicconditions for 1-4 days to develop photopigments (e.g., chlorophyll,carotenoids), lipids, proteins, and other compounds using thephototrophic metabolism. The time period of culturing phototrophic andmixotrophic stages may be species specific and dependent on growth rateor contamination risks.

Cultures may then be harvested in their entirety or harvested partially(i.e., 10%-90% harvest) and then transitioned to be cultured inmixotrophic conditions to increase growth. The process may be repeatedamong multiple series of bioreactors. The process allows for finalproduct curing while producing seed (i.e., inoculum) for the nextbioreactor. This process also allows for: reduced cost through less seedproduction; conditioning of microorganisms for outdoor production as itcompletes each stage of bioreactors; bioreactors which may reseedthemselves after a harvest; and modifying the water quality of theculture by eliminating organic carbon during phototrophic growth. Insome embodiments, bacteria that have been identified as providing apositive or neutral effect on the microorganism culture may be added inany one of or combination of stages for multiple purposes such asenhancement of the culture growth, health, and product formation.

Transitions Between Heterotrophy and Mixotrophy

A culture of heterotrophic microorganisms may be axenically produced infermenters (i.e., heterotrophic bioreactors) at a large scale usingconvention fermentation technology. The heterotrophically producedmicroorganisms may be used to inoculate a mixotrophic or phototrophicculture. The utilization of bacterial free or low-bacteria (<10⁴cells/mL) inoculum may help prevent the colonization of contaminatingbacteria in non-axenic mixotrophic cultures in larger production-scalereactors (>500 L volume). Additionally, it has been observed in somespecies that when phototrophically cultured microalgae is utilized toinoculate a mixotrophic culture, the mixotrophic culture may experiencea longer lag period (e.g., 1-100 hours) in the initial growth thanmicroalgae which was previously cultured with at least some organiccarbon due to the stress experienced by the cells in phototrophicculturing conditions. Therefore, using heterotrophically culturedmicroalgae as inoculum for a mixotrophic culture may mitigate the growthlag experienced in mixotrophic cultures inoculated with phototrophicallycultured inoculum. In some embodiments, a culture of microorganisms maybe grown axenically in a fermenter vessel to a high density in a firststep and then seeded in large outdoor bioreactors that are open andcapable of operation in phototrophic and mixotrophic culture conditions.

Example 2

Haematococcus is known to produce Astaxanthin, with the concentration ofAstaxanthin within the cells increasing when the Haematococcus cells arecultured in conditions with at least some light. Most of theChlorophyceae class of microalgae have developed a phototrophicmetabolism, but also evolved developing a heterotrophic metabolism thatmay be capable of growth in the absence of light with a source oforganic carbon.

In one non-limiting embodiment illustrating the potential use ofheterotrophy to produce axenic seed for a phototrophic or mixotrophicculture, a fermentation vessel may be utilized to create axenicHaematococcus seed for large outdoor open pond bioreactors reactorsreceiving at least some light. As shown in FIG. 4, a fermentation vesselmay comprise tubing, air filters, media filter, a sampling devise andquick connectors that will be sterilized in an autoclave and carried toa flow hood for preparation. The media will be sterilized throughfiltration by pumping the media into the fermentation vessel through a0.1 micron sterile filter. The inlet air will also be sterilized beforeentering the fermentation vessel. After media supply, the fermentationvessel will be inoculated with an axenic Haematococcus culture grown inflasks. Glucose, sucrose, acetic acid, and/or sodium acetate will beused as the organic carbon source in the fermentation vessel.

The experimental data in FIG. 5 shows that a fermentation vesselutilizing an organic carbon feed of sodium acetate for Haematococcuswill reach a concentration of 600,000 cells/ml and will be ready to betransfer the culture of Haematococcus to a larger culturing vessel, suchas an open outdoor pond. If the fermentation is not supplied with morenutrients the Haematococcus cells may encyst. The system may need to bereplenished with more acetate and nutrients to get higher celldensities. Without an additional batch of organic carbon the cells willstart to encyst on about the third day of growing due to a depletion ofnutrients and a low dissolved oxygen level. A low rate of mixing withair will be used as Haematococcus may not be able to handle high ratesof mixing with air. A fermentation vessel with a higher capacity of masstransfer through the supply of more dissolved oxygen and mixing, alongwith more nutrient addition, may produce higher cell densities of 2 to2.5 million cells/ml or 1.5 g/1 of biomass. The higher cell densitieswill allow for the utilization of larger inoculums in open outdoor pondsfor mixotrophic or phototrophic culturing. The Astaxanthin whichaccumulates in the Haematococcus cells in the mixotrophic orphototrophic culture phase will be extracted from the Haematococcuscells using any known method of pigment extraction such as, but notlimited to, solvent extraction and supercritical carbon dioxideextraction.

In another embodiment, a culture of mixotrophically or phototrophicallyproduced microorganisms may inoculate a heterotrophic culture. Themixotrophically or phototrophically produced culture inoculating aheterotrophic culture may provide a targeted product in themicroorganism or a higher culture density in a shorter time period. Aswas discussed above with mixotrophic and phototrophic cultures,mixotrophic and heterotrophic cultures may also accumulate pigments(e.g., chlorophyll, carotenoids), proteins, lipids, and other productsdifferently in the microorganism depending on the culturing methods,which allows for the culturing methods to target selected products.

Example 3

Haematococcus is known to produce Astaxanthin in culturing conditionswith at least some light. The Astaxanthin production is typicallyinduced in nitrogen limited cultures and under high light intensity. Theprocess for Astaxanthin production from Haematococcus in phototrophicconditions is time consuming and requires a large physical footprint toallocate the relatively low cell density of the culture for exposure tothe light (e.g., short light path in shallow raceway ponds).

Alternatively, as shown in FIG. 6, the increase in red cysts after 96hours demonstrates the Astaxanthin accumulation phase may be inducedunder heterotrophic conditions using the smaller physical footprint of afermentation vessel (as compared to a short light path of shallowraceway ponds). Once the biomass of Haematococcus is produced in pondsphototrophically over a period of 1-7 days, the cells will beconcentrated between 3 and 500 times, and transferred to a fermentationvessel (i.e., heterotrophic bioreactor) that will be supplied with anorganic carbon source as an energy and carbon source. Thecarotenogenesis process which forms the Astaxanthin will occur in thefermentation vessel under strictly heterotrophic conditions. The cellswill then increase their formation of pigments and the number of redcysts over time (i.e., the heterotrophic induction phase). The culturesusing a fermentation vessel will have a foot print approximately 85times smaller than the phototrophic ponds used in the “reddening” orcyst cell formation stage ponds.

Transitions Between Phototrophy, Mixotrophy, and Heterotrophy

The transitioning of a culture of microorganisms, such as microalgae andcyanobacteria, between culture conditions may comprise controlling lightexposure, carbon dioxide supply, and organic carbon supply for theculture. When transitioning to phototrophy: the exposure of the cultureof microorganisms to light may be increased to a high light level, thesupply of carbon dioxide to the culture may be increased to a higherlevel, and the supply of organic carbon will cease. When transitioningto mixotrophy: the exposure of the culture of microorganisms to lightmay be increased (i.e., transition from heterotrophy) or decreased(i.e., transition from phototrophy) to a low light level, the supply ofcarbon dioxide may be increased (i.e., transition from heterotrophy) ordecreased (i.e., transition from phototrophy), and the organic carbonsource will be supplied. When transitioning to heterotrophy: theexposure of the culture of microorganisms to light will cease, thesupply of carbon dioxide will cease, and the organic carbon source willbe supplied. In some embodiments, different wavelengths, photoperiods,and intensities of light may be applied in phototrophic and mixotrophicconditions, such as but not limited to monochromatic (single lightwavelengths) light wavelengths between 200-800 nm (e.g., redwavelengths, blue wavelengths), combinations of various lightwavelengths and intensities of light wavelengths between 200-800 nm,constant light, and flashing light. In some embodiments high lightlevels may comprise light above 400 μmol/m² s, and low light levels maycomprise light below 400 μmol/m² s, however the sensitivity to andutilization efficiency of light will vary between species, and thus highand low light levels may be species specific.

In some embodiments, a culture may transition from phototrophy tomixotrophy to heterotrophy. In some embodiments, a culture maytransition from heterotrophy to mixotrophy to phototrophy. In someembodiments, a culture may transition from phototrophy to heterotrophyto mixotrophy. In some embodiments, a culture may transition frommixotrophy to phototrophy to heterotrophy. In some embodiments, aculture may transition from mixotrophy to heterotrophy to phototrophy.In some embodiments, a culture may transition from heterotrophy tophototrophy to mixotrophy. In some embodiments, the transitions of theculture of microorganisms between culture conditions may be cyclic orfollow a pattern.

Transition Based on Sunlight Levels

In some embodiments, the transition between trophic conditions maycorrespond to the light/dark cycle of the sun. Phototrophic cultures ofmicroorganisms may lose growth achieved during the high light conditionsof daylight through respiration as the availability of light is limitedduring the night and hours of low sunlight during sunrise and sunset.The use of artificial lights to provide 24 hour lighting to maintainphototrophic growth may add costs to the system and reduce the overallenergy efficiency of the microorganism culturing system, which isundesirable in a commercial setting. However, transitioning the cultureto mixotrophy in the low light portions of sunset and heterotrophyduring the night (i.e., absence of light) may preserve the growthachieved through phototrophy during the day, and possibly add additionalgrowth. In some embodiments, the culture may be transitioned fromheterotrophy to mixotrophy in the low light conditions of sunrisefollowing night time, and to phototrophy during the high lightconditions of daylight. In some embodiments, the culture may transitionfrom phototrophy to mixotrophy during the low light conditions of sunset following the high light conditions of daylight, and to heterotrophyduring night time.

In some embodiments, the culture of microorganisms may be fed organiccarbon outside of the peak hours of sunlight, and no organic carbonduring the peak hours of sunlight. The peak hours of sunlight defined asan equal amount of time on either side of the time when the sun is atits zenith. In some embodiments, the peak hours of sunlight may comprise6-10 hours. Night may be defined as the time period of no sunlightbetween when the sun has completely set and when the sun begins to rise.In some embodiments, a culture may transition from phototrophy tomixotrophy to heterotrophy in the time frames corresponding to the peakhours of sunlight (phototrophy), the non-peak hours of sunlight(mixotrophy), and night (heterotrophy). In some embodiments, a culturemay transition from heterotrophy to mixotrophy to phototrophy in thetime frames corresponding to night (heterotrophy), the non-peak hours ofsunlight (mixotrophy), and the peak hours of sunlight (phototrophy). Insome embodiments, a culture may transition from phototrophy tomixotrophy to heterotrophy to mixotrophy to phototrophy in a repeatingcycle based on the light/dark cycle of the sun and peak hours ofsunlight. In some embodiments, the organic carbon may be added at nightwhen the sun sets, and when the sun begins to rise organic carbon is nolonger added to the system. In some embodiments, the culture ofmicroorganisms may be cultured with a method using combinations ofphototrophic, mixotrophic, and heterotrophic culture conditions for atleast two consecutive days, and in preferred embodiments for 2 to 90consecutive days.

Transition Based on Threshold Condition

In some embodiments, the time a culture of microorganisms spends in aculture condition before transitioning to another culture condition maybe dictated by the occurrence of at least one threshold condition suchas, but not limited to, a detected level of culture cell density, adetected level of dissolved oxygen, a detected level of dissolved carbondioxide, a detected pH level, a detected level of contaminatingorganisms, a detected level of chlorophyll, complete consumption of abatch of nutrients, complete consumption of a batch of organic carbon, adetected temperature level, a detected residual nitrate level, and adetected residual organic carbon level. In some embodiments, thetransition to another culture condition may occur when the detectedlevel is below a threshold level or set point. In some embodiments, thetransition to another culture condition may occur when the detectedlevel is above a threshold level or set point.

In some embodiments, the transition from phototrophy to mixotrophy orheterotrophy may occur when the detected dissolved oxygen level is abovea threshold level. A transition from phototrophy to mixotrophy orheterotrophy in a high dissolved oxygen states allows the microorganismto consume some of the oxygen to reduce an inhibiting effect on themicroorganisms' growth that may occur in phototrophic culture conditionswith high dissolved oxygen. In some embodiments, the transition frommixotrophy or heterotrophy to phototrophy may occur when the detecteddissolved oxygen level is below a threshold level.

A transition from mixotrophy or heterotrophy, where the microorganismsconsume oxygen, to phototrophy may allow for the oxygen in the cultureto be replenished through photosynthetic activity instead of from anexternal source and enable further heterotrophic growth in a subsequentculturing stage. In some embodiments, the dissolved oxygen thresholdlevel may range from 0.1 mg O₂/L to about 30 mg O₂/L. In someembodiments, the dissolved oxygen level may be decreased to 6 mgO₂/L-0.1 mg O₂/L in phototrophic conditions to reduce potentialcontamination prior to switching to mixotrophic conditions. In someembodiments, the level of dissolved oxygen may be increased to 10 mgO₂/L-30 mg O₂/L to reduce contamination prior to switching the cultureto mixotrophic conditions.

In some embodiments, the transition from mixotrophy or heterotrophy tophototrophy may occur when the detected carbon dioxide level is above athreshold level or set point. Microorganisms in mixotrophic orheterotrophic culture conditions may produce carbon dioxide to asaturation point which slows the mixotrophic or heterotrophic growth,but may boost phototrophic growth where carbon dioxide is consumed. Thesaturation level of carbon dioxide that inhibits growth and consumptionrate of carbon dioxide may be species specific. In some embodiments, thetransition from phototrophy to mixotrophy or heterotrophy may occur whenthe detected carbon dioxide level is below a threshold level. Thetransition from phototrophy, wherein carbon dioxide is consumed, tomixotrophic or heterotrophic conditions of net carbon dioxide productionmay allow for the carbon dioxide level of the culture to be replenishedthrough metabolic activity instead of from an external source.Replenishing the carbon dioxide level may enable further photosyntheticgrowth in a subsequent culturing stage.

In some embodiments, the transition from heterotrophy to mixotrophy orphototrophy, or the transition from mixotrophy to phototrophy, may occurwhen the detected level of chlorophyll is below a threshold level or setpoint. Microorganisms in heterotrophic conditions comprising an absenceof light may produce less chlorophyll than cultures exposed to at leastsome light. The chlorophyll level in the microorganisms may be increasedby transitioning from heterotrophic to phototrophic or mixotrophicconditions comprising at least some light when the detected chlorophylllevel is low, or from mixotrophic to phototrophic for better utilizationof high light conditions when the chlorophyll level is low. In someembodiments, the transition from phototrophy or mixotrophy toheterotrophy may occur when the detected level of chlorophyll is above athreshold level.

While some level of chlorophyll may be desirable for certain productmarkets, transitioning the culture from phototrophy or mixotrophy toheterotrophy may maintain the chlorophyll at a desired level or reducethe chlorophyll content in a final culture stage. In some embodiments,the light exposure may initially comprise lower energy light (e.g.,wavelengths between 500 nm-800 nm) and/or lower levels of intensitiesslowly increased over time. In some embodiments, the light exposure mayinitially comprise higher energy light (e.g., wavelengths between 200nm-500 nm) and/or lower levels of intensities slowly increased overtime. In some embodiments, the light exposure may initially comprisehigher energy light (e.g., wavelengths between 200 nm-500 nm) and/orhigher levels of intensities slowly increased over time.

In some embodiments, the transition from phototrophy to mixotrophy mayoccur when the detected level of culture cell density is above a firstthreshold level or set point. The first threshold level may comprise aculture density at which self shading begins to inhibit growth, such asbut not limited to a density of 1 g/L for some species. In furtherembodiments, the transition from mixotrophy to heterotrophy may occurwhen the detected level of the culture density is above a secondthreshold level. The second threshold level may comprise a culturedensity at which the mixing regime is no longer capable of exposing allcells of the culture to at least some light, such as but not limited to2 g/L for some species.

A culture of microorganisms which starts out in phototrophic conditionsmay utilize the low cell culture density to minimize self-shading andmaximize exposure to light for photosynthetic activity. As the cellculture density increases to a first threshold level due to thephototrophic growth and with an accompanying increase in self-shading,the growth of the microorganism culture may be sustained bytransitioning to mixotrophic conditions which require less light and canalso supply energy to the microorganisms through an organic carbonsource. As the cell culture density increases to a second thresholdlevel due to the mixotrophic growth and with an accompanying increase inself-shading, the growth of the microorganism culture may be sustainedby transitioning to heterotrophic conditions which requires no light andsupplies energy to the microorganisms through an organic carbon source.The culture densities at which the growth stagnates for certain growthconditions may be species specific.

In some embodiments, the transition between culture conditions may occurwhen the pH level is below a threshold level or set point. In someembodiments, the transition between culture conditions may occur whenthe pH level is above a threshold level. In some embodiments, the pHlevel ranges from about 6 to about 9. In other embodiments, the pH levelranges from about 1 to about 5. The pH level of a culture may beinfluenced by a variety of factors in the culture such as, but nolimited to, the inorganic carbon, the organic carbon, and the metabolicactivity of the microorganism. Therefore, the pH level may be used as anindicator to initiate a transition to different culture conditions,which use different types or amounts of carbon and alters the metabolicactivity of the microorganism to maintain the microorganisms in optimalculture conditions for growth or product accumulation.

In some embodiments, the transition from phototrophy to mixotrophy orheterotrophy may occur when the detected level of contaminatingorganisms is below a threshold level or set point. The organic carbonprovided in mixotrophic and heterotrophic conditions may provide a feedsource for contaminating organisms, and thus allowing the transition tomixotrophy or heterotrophy from phototrophy only when the detected levelof contaminating organisms is low may keep the contaminating organismspopulation under control. In some embodiments, the transition frommixotrophy or heterotrophy to phototrophy may occur when the detectedlevel of contaminating organisms is above a threshold level. Thetransition to phototrophy may reduce the organic carbon available asfeed to the contaminating organisms and help keep the contaminatingorganism population under control. In some embodiments, the desiredlevel of contaminating organisms is less than 25%, 20%, 10%, or 5% ofthe total cells in a culture. Methods of detecting contaminatingorganisms may comprise any method known in the art such as, but notlimited to, observation of a sample under a microscope, automatednucleic acid sequencing, oil immersion-phase contrast micrograph andcell cytometry with bacterial dying, and quantitative polymerase chainreaction (QPCR).

In some embodiments, the transition from phototrophy to mixotrophy orheterotrophy may occur when the detected residual organic carbon levelis below a threshold level or set point. The organic carbon provided inmixotrophic and heterotrophic conditions may provide a feed source forcontaminating organisms, and thus allowing the transition to mixotrophyor heterotrophy from phototrophy only when the detected residual organiccarbon level is low may keep the contaminating organisms populationunder control by only having enough organic carbon in the culture forthe microalgae or cyanobacteria to consume. In some embodiments, thetransition from mixotrophy or heterotrophy to phototrophy may occur whenthe detected residual organic carbon level is above a threshold level.The transition to phototrophic conditions with no organic carbon feedmay reduce the organic carbon available as feed to the contaminatingorganisms and help keep the contaminating organism population undercontrol. In some embodiments, the detection of other residual nutrients(e.g., nitrates) is a trigger for when to transition between cultureconditions may be used in a similar as that described above for organiccarbon to achieve the goal of minimizing the feed source available tocontaminating organisms. In some embodiments, the level of residualorganic carbon and other nutrients may comprise less than 1,000 ppm, 500ppm, 250 ppm, 200 ppm, 100 ppm, or 50 ppm. Consumption rate of organiccarbon, nitrates, or other nutrients may be species specific.

Similarly, the transition between culture conditions may also bedictated by consumption of a batch of nutrients, nitrates, and/ororganic carbon. In some embodiments, the culture of microorganisms maytransition from mixotrophy or heterotrophy to phototrophy when a batchof organic carbon has been completed consumed. The complete consumptionof a batch of nutrients, nitrates, and organic carbon may be indicatedby a detection of residual nutrients, nitrates or organic carbon anegligible amounts (e.g. 25 ppm or less).

In some embodiments, the transition from heterotrophy to mixotrophy orphototrophy may occur when the temperature of the culture is below athreshold level or set point. In some embodiments, the transition fromphototrophy to mixotrophy or heterotrophy may occur when the temperatureof the culture is above a threshold level. In some embodiments, thetemperature of the culture ranges from about 10° C. to about 30° C. Inother embodiments, the temperature of the culture ranges from about 30°C. to about 50° C. The temperature of a culture may be influenced by avariety of factors in the culture such as, but no limited to, the lightand the metabolic activity of the microorganism. A transition fromheterotrophy (i.e., no light) or mixotrophy (i.e., low light) tophototrophy (i.e., high light) may increase the temperature of theculture due to the heat from the supplied light energy. A transitionfrom phototrophy (i.e., high light) to heterotrophy (i.e., no light) ormixotrophy (i.e., low light) may decrease the temperature of the culturedue to the heat from the supplied light energy. Therefore, thetemperature of a culture may be used as an indicator to initiate atransition to different culture conditions in which different amounts oflight may influence the culture temperature and alter the metabolicactivity of the microorganism.

When transitioning a culture between trophic conditions based onsunlight levels or threshold conditions, the transition may be automatedby parameter detection equipment (e.g., probes, sensors), a computer,and programmable logic controller (PLC) receiving input from parameterdetection equipment and controlling valves and actuators. In someembodiments, a photodiode or photodetector and photovoltaic panel may beused in combination to transition a culture from phototrophic tomixotrophic or heterotrophic conditions in an automated manner. Thephotodiode or photodetector may detect light that is below a thresholdvalue or set point which indicates low light conditions such as, but notlimited to, cloudy days, rainy days, non-peak sunlight hours, and night.When light is detected below the threshold value, the PLC may transitionthe culture from phototrophic culture conditions to mixotrophic orheterotrophic conditions by controlling the aspects of the system suchas, but not limited to, light supply/exposure, inorganic carbon supply,organic carbon supply, dissolved oxygen, and mixing.

In some embodiments, the organic carbon supply rate and the inorganiccarbon supply rate may be monitored and controlled by the PLC tomaintain the culture conditions in a net carbon dioxide usage state asthe PLC transitions the culture between conditions over a time period.Maintaining a net carbon dioxide usage state may be useful in carbonremediation applications of microorganism cultures. In some embodiments,a dissolved oxygen (DO) probe may be used as a proxy for a light orphoto sensors. The DO probe may monitor the DO concentration, and enablethe inference by the PLC that when the DO level is rising the light maybe available for phototrophic conditions and initiate a transition tophototrophy. Also, when the DO level stops rising, light may not beavailable for phototrophic conditions and the PLC may initiate atransition to mixotrophic or heterotrophic conditions.

In some embodiments, a culture operating under phototrophic conditionsutilizing carbon dioxide as inorganic carbon may transition tomixotrophic conditions with the addition of an organic carbon source inthe bioreactor. The organic carbon source may be added in a cyclicalpattern spaced in minutes to hours via feedback control from a detectedculture parameter (e.g., pH, nitrate, total organic carbon, opticaldensity, dissolved oxygen, bacteria contamination). Such cultureparameters may be measured and monitored by probes and sensors known inthe art and available commercially from companies such as YSI.

In some embodiments, the organic carbon may be added in a batch methodwhere a particular amount of the organic carbon is added to supportabout 24 hours of growth (dependent on species), then the bioreactor mayrun for about 24 hours in phototrophic conditions with no organiccarbon, and this process may be repeated. In some embodiments, theorganic carbon may be added in a method where mixotrophic conditionsoperate for about 7-10, transitioned to phototrophic conditions forabout 2 days, and then transitioned back to mixotrophic conditions. Themethods described for adding organic carbon in this specification may beused individually or in combination.

Bioreactor Systems with Multiple Zones

In some embodiments, the transition between culture conditions may benot be dictated solely by culture parameters, but instead may also bedictated by the configuration of a culturing vessel or bioreactorsystem. In some embodiments, a bioreactor system may have organic carbonsupply devices disposed in some portions and no organic carbon supplydevices in other portions. In some embodiments, a bioreactor system mayprovide varying levels of light exposure to the culture of microalgae insome portions and block all exposure to light in other portions. In someembodiments, a bioreactor system may have carbon dioxide supply devicesdisposed in some portions and no carbon dioxide supply devices in otherportions.

By circulating the microorganism culture through portions of abioreactor system with different combinations of organic carbon supplydevices, carbon dioxide supply devices, and exposure to light, themicroorganism culture may be transitioned between phototrophic,mixotrophic, and heterotrophic zones of culture conditions. In someembodiments, at least one phototrophic culture zone may comprise atleast one carbon dioxide supply device, no organic carbon supply device,and may be configured to expose the culture of microorganisms to atleast some light. In some embodiments, the phototrophic zone maycomprise an organic carbon supply device which may be deactivated duringphototrophic culture conditions. In some embodiments, at least onemixotrophic culture zone may comprise at least one carbon dioxide supplydevice, at least one organic carbon supply device, and may be configuredto expose the culture of microorganisms to at least some light. In someembodiments, at least one heterotrophic culture zone may comprise anorganic carbon supply device, no carbon dioxide supply device, and maybe configured to block the microorganisms from exposure to light. Insome embodiments, the bioreactor system may be configured to allow themicroorganisms to consume substantially all organic carbon supplied tothe microorganisms in a mixotrophic or heterotrophic zone before theculture of microorganisms enter a phototrophic zone.

In some embodiments, a circulation system such as, but not limited to,at least one pump and fluid conduit may circulate the culture throughoutthe bioreactor system and plurality of zones. In some embodiments, thecirculation of the microorganism culture between the at least onephototrophic, mixotrophic, and heterotrophic zones may be dictated by adetected culture parameter such as, but not limited to, cell culturedensity, dissolved oxygen, dissolved carbon dioxide, pH, contamination,chlorophyll content, nutrient levels, and temperature. In someembodiments the bioreactor system may comprise adjustable artificiallights and light blocking devices (e.g., louvers, covers, awnings,photovoltaic panels), controllable organic carbon supply devices, andcontrollable carbon dioxide supply devices, which may transition asingle zone in the bioreactor system between functions as aphototrophic, mixotrophic, and heterotrophic zone.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A method of culturing microorganisms, comprising:a. Providing a culture of microorganisms in an aqueous culture mediumcapable of growth on light as an energy source, carbon dioxide as acarbon source, and at least one organic carbon source as an energy andcarbon source; b. Transitioning the culture of microorganisms between atleast two culture conditions selected from the group consisting ofphototrophic, mixotrophic, and heterotrophic culture conditions, atleast once in the life of the culture of microorganisms, wherein: i.Phototrophic culture conditions comprise a supply of light, a supply ofcarbon dioxide, and the absence of a supply of an organic carbon source;ii. Mixotrophic culture conditions comprise a supply of light, and asupply of at least one organic carbon source; iii. Heterotrophic cultureconditions comprise a supply of at least one organic carbon source andthe absence of a supply of light; c. Wherein the transition between theat least two culture conditions selected from the group consisting ofphototrophic, mixotrophic, and heterotrophic culture conditions occurswhen at least one threshold condition of the culture is met, the atleast one threshold condition comprising: i. A detected level ofdissolved oxygen; ii. A detected level of chlorophyll; iii. A detectedlevel of cell culture density; and iv. A detected level of light.
 2. Themethod of claim 1, wherein the culture of microorganisms comprises atleast one selected from the group consisting of microalgae andcyanobacteria.
 3. The method of claim 2, wherein the microalgaecomprises Chlorella.
 4. The method of claim 1, wherein the at least oneorganic carbon source is selected from the group consisting of acetate,acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid,butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids,galactose, glucose, glycerol, glycine, lactic acid, lactose, maleicacid, maltose, mannose, methanol, molasses, peptone, plant basedhydrolyzate, proline, propionic acid, ribose, sacchrose, partial orcomplete hydrolysates of starch, sucrose, tartaric, TCA-cycle organicacids, thin stillage, urea, industrial waste solutions, and yeastextracts.
 5. The method of claim 1, wherein the transition betweenculture conditions is automated by a computer and programmable logiccontroller receiving input from the at least one sensor detecting the atleast one threshold condition to control at least one of the supply oflight, supply of carbon dioxide, and supply of the at least one organiccarbon source.
 6. The method of claim 1, wherein the culture ofmicroorganisms transitions from phototrophic to mixotrophic cultureconditions when the detected dissolved oxygen level is above a thresholdlevel.
 7. The method of claim 1, wherein the culture of microorganismstransitions from phototrophic to heterotrophic culture conditions whenthe detected dissolved oxygen level is above a threshold level.
 8. Themethod of claim 1, wherein the culture of microorganisms transitionsfrom mixotrophic to phototrophic culture conditions when the detecteddissolved oxygen level is below a threshold level.
 9. The method ofclaim 1, wherein the culture of microorganisms transitions fromheterotrophic to phototrophic culture conditions when the detecteddissolved oxygen level is below a threshold level.
 10. The method ofclaim 1, wherein the culture of microorganisms transitions fromheterotrophic to mixotrophic culture conditions when the detectedchlorophyll level is below a threshold level.
 11. The method of claim 1,wherein the culture of microorganisms transitions from heterotrophic tophototrophic culture conditions when the detected chlorophyll level isbelow a threshold level.
 12. The method of claim 1, wherein the cultureof microorganisms transitions from mixotrophic to phototrophic cultureconditions when the detected chlorophyll level is below a thresholdlevel.
 13. The method of claim 1, wherein the culture of microorganismstransitions from phototrophic to mixotrophic culture conditions when thedetected chlorophyll level is above a threshold level.
 14. The method ofclaim 1, wherein the culture of microorganisms transitions fromphototrophic to heterotrophic culture conditions when the detectedchlorophyll level is above a threshold level.
 15. The method of claim 1,wherein the culture of microorganisms transitions from phototrophic tomixotrophic culture conditions when the detected level of cell culturedensity is above a threshold level.
 16. The method of claim 1, whereinthe culture of microorganisms transitions from mixotrophic toheterotrophic culture conditions when the detected level of cell culturedensity is above a threshold level.
 17. The method of claim 1, whereinthe culture of microorganisms transitions from heterotrophic tomixotrophic culture conditions when the detected level of cell culturedensity is above a threshold value.
 18. The method of claim 1, whereinthe culture of microorganisms transitions from heterotrophic tophototrophic conditions when the detected level of cell culture densityis above a threshold value.
 19. The method of claim 1, wherein theculture of microorganisms transitions from phototrophic to mixotrophicculture conditions when the detected level of light is below a thresholdlevel.
 20. The method of claim 1, wherein the culture of microorganismstransitions from phototrophic to heterotrophic culture conditions whenthe detected level of light is below a threshold level.